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Keywords:

  • ammonium toxicity;
  • eutrophication;
  • heathlands;
  • internal acidification;
  • matgrass swards;
  • nitrogen deposition;
  • survival

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • The effects of increasing ammonium concentrations in combination with different pH levels were studied on five heathland plant species to determine whether their occurrence and decline could be attributed to ammonium toxicity and/or pH levels.
  • Plants were grown in growth media amended with four different ammonium concentrations (10, 100, 500 and 1000 µmol l−1) and two pH levels resembling acidified (pH 3.5 or 4) and weakly buffered (pH 5 or 5.5) situations.
  • Survival of Antennaria dioica and Succisa pratensis was reduced by low pH in combination with high ammonium concentrations. Biomass decreased with increased ammonium concentrations and decreasing pH levels. Internal pH of the plants decreased with increasing ammonium concentrations. Survival of Calluna vulgaris, Deschampsia flexuosa and Gentiana pneumonanthe was not affected by ammonium. Moreover, biomass increased with increasing ammonium concentrations. Biomass production of G. pneumonanthe reduced at low pH levels.
  • A decline of acid-sensitive species in heathlands was attributed to ammonium toxicity effects in combination with a low pH.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In Western Europe, heathlands and matgrass swards were once common vegetation types (De Smidt, 1975; Gimingham, 1992). From the mid-eighteenth century, however, European heathlands declined dramatically in area, mainly as a result of reclamation for forestry and farming (De Smidt, 1975, 1979; Webb, 2002). Their unique composition of many characteristic and endangered plant and animal species makes these ecosystems subject to conservation and restoration (Gimingham, 1992; Webb, 1998).

In addition to losses through habitat destruction, the biodiversity of dry and wet heathlands and related matgrass swards is rapidly declining (Roelofs et al., 1996; Bobbink et al., 1998). Plant species diversity in heathlands in the Netherlands has declined over 50% in the past 50 yr because of environmental stresses (Vonk et al., 2001), and many herbaceous species such as Antennaria dioica, Arnica montana, Cirsium dissectum, and Gentiana pneumonanthe have largely disappeared and are seriously threatened. Conversely, grasses such as Deschampsia flexuosa and Molinia caerulea have become dominant.

It was suggested that this shift towards grass-dominated vegetation was caused by the increased atmospheric deposition of nitrogen- and sulphur-containing compounds during the past decades, resulting in eutrophication and acidification of the soil (Heil & Diemont, 1983; Bobbink et al., 1998). Earlier studies (Heil & Diemont, 1983; Berendse & Aerts, 1984; Roelofs, 1986; Aerts et al., 1990) showed that grasses had higher productivity at elevated N concentrations compared with herbaceous species and shrubs. Grasses were therefore thought to outcompete herbaceous species and shrubs at high N availability (Aerts & Berendse, 1988; Pitcairn et al., 1991). However, a study by Houdijk et al. (1993) showed that many herbaceous species had already disappeared from the heathlands before grasses became dominant, indicating that processes other than competition for light and nutrients play an important role in the decline of herbaceous species.

Nitrogen deposition in the Netherlands is mainly in the form of ammonium (NH4+) (Erisman, 1990; Bobbink et al., 1992; Boxman et al., 1998; Kreutzer et al., 1998) and, as a result, plants encounter increased NH4+ concentrations which have been shown to be toxic for many plant species (Mehrer & Mohr, 1989; Britto & Kronzucker, 2002 and references therein), including herbaceous heathland species such as A. montana and C. dissectum (De Graaf et al., 1998; Dorland et al., 2003; Lucassen et al., 2003).

In addition, the atmospheric deposition of S and N results in acidification of heathland soils and the depletion of buffering base cations such as Ca2+, Mg2+ and K+ (Carnol et al., 1997). This may lead to a change in soil buffering and the loss of acid-sensitive species (Houdijk et al., 1993). For degraded, acidified, dry heathlands an average soil pH of 3.8–4.2 was found, whereas for degraded, acidified, wet heaths a slightly higher pH of 4.0–4.5 (caused by reduction processes) was measured (De Graaf et al., 1994; Roelofs et al., 1996; Dorland et al., 2003). Acidification in wet heaths is also induced by lowering of groundwater tables, as this enhances acidifying processes such as the oxidation of iron sulphides, mineralization and the influence of acidic rainwater (Roelofs, 1993; Grootjans et al., 1996; Runhaar et al., 1996; Lamers et al., 1998). Because of the acid soil conditions, nitrification in heathland soils is low and is strongly reduced with decreasing pH (Roelofs et al., 1985; Van Breemen & Van Dijk, 1988; Dorland et al., 2004). As nitrification is an acidifying process, the inhibition of nitrification constitutes a negative feedback on acidification. All these processes contribute directly or indirectly to an accumulation of NH4+ in the soil, mostly accompanied by higher NH4+ availabilities.

Although external pH was suggested as the primary cause of the decline of herbaceous species in favour of grasses (Van Dam et al., 1986; Dueck & Elderson, 1992; Houdijk et al., 1993), others found no effects of external pH on the growth of herbaceous heathland species (Van Dobben, 1991), or found indirect pH effects through aluminium toxicity (Heijne et al., 1996). NH4+ concentrations did not explain the dramatic decline in plant diversity either, as many herbaceous species were shown to grow well on high NH4+ concentrations under weakly buffered conditions (Bobbink et al., 2003).

The relationship between NH4+ toxicity and soil acidification has been the subject of a number of studies (Findenegg, 1987; Dijk & Eck, 1995; Dijk & Grootjans, 1998; Lucassen et al., 2003). Lucassen et al. (2003) suggested that the decline of C. dissectum was caused by the combination of high NH4+ concentrations and a low pH of the growth medium. They hypothesized that at high NH4+ concentrations and low external pH, C. dissectum suffered from low internal pH levels as a result of reduced proton excretion. This is explained by proton excretion which was found to occur when NH4+ is assimilated in plants (Raven & Smith, 1976; Findenegg, 1987; Van Beusichem et al., 1988; Goodchild & Givan, 1990). Others also found that growth on NH4+ results in a decrease in tissue pH (Gerendás et al., 1990; De Graaf et al., 2000).

In this study we describe the results of a hydroponic experiment with five heathland species from both wet and dry heaths: G. pneumonanthe, Succisa pratensis, Calluna vulgaris, A. dioica and D. flexuosa. Plants were subjected to environmental stress by growing them in media with different NH4+ concentrations and pH levels. Biomass, mortality, internal pH of plants and the chemical composition of plants were measured to estimate the fitness and survival of plants. NH4+-tolerant species are usually found in acidic habitats and are likely to be adapted to NH4+ nutrition, as NH4+ is the dominant N form at low pH (Gigon & Rorison, 1972; Troelstra et al., 1990). In contrast, species from less acidic habitats usually prefer nitrate (Falkengren-Grerup & Lakkenborg-Kristensen, 1994; Britto & Kronzucker, 2002). Therefore we hypothesized that acidification would negatively affect the survival and fitness of A. dioica and S. pratensis as these are characteristic for weakly buffered conditions and are regarded as acid-sensitive species. It was also expected that these effects will be enhanced by increased NH4+ concentrations. Calluna vulgaris and D. flexuosa can be found in eutrophied and acidified heathlands and are therefore thought to be more acid-tolerant. These species were expected to be less affected by low external pH in combination with high NH4+ concentrations. The rare herbaceous species G. pneumonanthe is regarded as slightly acid-tolerant and thus less susceptible to low external pH than A. dioica and S. pratensis.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant species

Antennaria dioica (L.) Gaertner, Gentiana pneumonanthe (L.) and Succisa pratensis (L.) Moench are perennial herbaceous species characteristic for species-rich, nutrient-poor matgrass swards and heathlands. In the Netherlands, A. dioica can be found on the dry parts of nutrient-poor heathlands and species-rich grasslands. Gentiana pneumonanthe is mainly found on wet heaths and species-rich grasslands. Both species are rare and threatened, and their present occurrence in the Netherlands is restricted to a few areas. Succisa pratensis is a more widespread species, found on various types of nutrient-poor grasslands such as matgrass swards and species-rich heathlands. Its distribution has been reduced by almost 75% since 1935 and it has been on the red list since 2002 (Van der Meijden, 2002). Calluna vulgaris Hill is a common shrub, characteristic for dry heaths and often dominating in species-poor heaths. Its distribution is widespread in Western Europe. In deteriorating heathlands suffering from acidification and eutrophication, C. vulgaris is replaced by grasses such as Deschampsia flexuosa (L.) Trin., a common grass which mainly occurs in N-enriched matgrass swards and heathlands, where it can reach cover percentages up to 100%.

Experimental design

Seedlings of A. dioica, G. pneumonanthe and S. pratensis were obtained by germinating seeds on nutrient-poor substrate (vermiculite). The seedlings were grown for 3–5 wk before the start of the experiments. Seeds of G. pneumonanthe and S. pratensis were collected in a Dutch nature reserve (Havelte-Oost, 52°48′ N, 6°13′ E); A. dioica seeds were ordered from Blauetikett-Bornträger GmbH, Offstein, Germany. Seedlings of C. vulgaris and D. flexuosa were collected in a heathland near Nijmegen (Mulderskop, 51°47′ N, 5°53′ E) and grown on vermiculite for 2 wk before the start of the experiment. In order to reduce initial differences between plants, plants of approximately the same size were selected.

Plants were carefully placed in polystyrene trays and transferred into 2 l opaque containers which were continuously aerated to prevent anoxic conditions. The seedlings were exposed to eight different growing solutions differing in NH4+ concentration and pH level. The NH4+ concentrations were 10, 100, 500 and 1000 µmol l−1. The pH levels resembled those of acidified and weakly buffered soil conditions of dry and wet heaths, respectively. Therefore G. pneumonanthe and S. pratensis were subjected to pH values of 4 (acidified) and 5.5 (weakly buffered), whereas A. dioica, C. vulgaris and D. flexuosa were subjected to pH values of 3.5 (acidified) and 5 (weakly buffered) (Table 1). The number of plants in each container was three for S. pratensis; four for G. pneumonanthe; seven for D. flexuosa and A. dioica; and eight for C. vulgaris.

Table 1.  NH4+ amendments and pH ranges for each species
SpeciespH rangeNH4+ concentration (µmol l−1)
low pHhigh pH
Gentiana pneumonanthe45.510–100–500–1000
Succisa pratensis45.510–100–500–1000
Antennaria dioica3.5510–100–500–1000
Calluna vulgaris3.5510–100–500–1000
Deschampsia flexuosa3.5510–100–500–1000

We added low amounts of nutrients to the culture media in order to represent natural conditions according to De Graaf et al. (1994). The concentrations of nutrients were 100 µmol l−1 Ca2+, 100 µmol l−1 Mg2+, 200 µmol l−1 K+, 100 µmol l−1 SO42–, 200 µmol l−1 PO43–, 0.27 µmol l−1 Fe, 0.7 µmol l−1 Zn2+, 0.8 µmol l−1 Mn2+, 0.2 µmol l−1 Cu2+, 0.008 µmol l−1 Mo, 0.8 µmol l−1 H3BO3. Fe was added as Fe-EDTA. 1-cyanguanidine was added to the culture media in a concentration which was 1% of the molar concentration of NH4+ to prevent nitrification. Nitrate concentrations were low throughout the experiment, indicating that nitrification was successfully reduced by the application of 1-cyanguanidine and the relative high flow velocity of medium through the trays. Growth media in the containers were continuously refreshed using a continuous flow system. Per container, 12.5 l medium was pumped weekly from 25 l reservoirs.

The pH of the growth solutions was set using 1 mmol l−1 HCl and 1 mmol l−1 NaOH. The pH of the growth solutions was checked every 2 d and adjusted when necessary (data not shown). The experiments were performed in a climate chamber with a day : night regime of 16 : 8 h and a temperature of 20 and 17°C, respectively. The humidity was 50–70% and the irradiance was 200 µmol m−2 s−1.

Survival and growth

During the experiment, mean survival per container was determined by measuring the percentage of plants that died weekly. Deschampsia flexuosa, G. pneumonanthe and S. pratensis were harvested after 8 wk, whereas A. dioica was harvested after 6 wk. The experimental period for C. vulgaris was 14 wk, as it is a slow growing species and visible effects of treatments were expected to occur later. After harvesting, plants were pooled per container and separated into shoots and roots. Total dry weight (biomass) was determined after drying the plant material for 24 h at 70°C.

Internal pH and nutrient composition

In order to measure the internal pH of the plants, plants were pooled per container and mixed thoroughly. Then 0.1–0.2 g of the mixed plant material was ground with 1 or 2 ml demineralized water, respectively, in a glass-to-glass Potter homogenizing device. A Sentron pH sensor (Sentron Europe BV, Roden, the Netherlands) in combination with a Sentron 1001 pH meter was used to measure pH in the Potter tube directly.

From the dried plants of G. pneumonanthe, C. vulgaris and D. flexuosa, root and shoot material was sampled and ground using liquid N, and 100 mg of plant material was digested in sealed Teflon vessels in a Milestone destruction microwave oven (MLS 1200 mega; Milestone, Shelton, CT, USA) with nitric acid and hydrogen peroxide. Plant material was analysed for calcium, magnesium, phosphorus, manganese and sulphur using an inductively coupled plasma emission spectrophotometer (ICP, Spectroflame Flame VML2; Spectro AI, Kleve, Germany). Potassium and sodium were determined with a flame photometer, using a Technicon I Auto Analyser (Technicon, New York, USA). To obtain a representative sample of carbon and N concentrations, two subsamples per sample were analysed using a Carlo Erba Na 1500 CNS analyser (Thermo Electron, Milan, Italy). The two subsamples were pooled for statistical analysis. Variation among the subsamples was small; standard deviations were at most 5% of the means for both C and N concentrations. The nutrient composition and C and N content of A. dioica and S. pratensis could not be analysed because of a lack of material, which was caused by the high mortality and small size of the plants.

Statistical analysis

All data were analysed using the spss 11.5 package (SPSS Inc., Chicago, IL, USA) after testing for normality. Mean survival per container was analysed using a general linear model (GLM) procedure. Survival was monitored regularly, but only final survival data were included in the statistical analysis, as we were mainly interested in the effects of the treatments on total survival. Survival percentages were arcsin of square root transformed on the survival factor (percentage/100).

The effects of NH4+ and external pH levels on shoot and root biomass, internal pH and internal chemical composition were analysed using a GLM procedure. Because of the high mortality in some of the treatments as opposed to low mortality (high survival) in other treatments, data on A. dioica and S. pratensis were unbalanced. Although mean biomass and mean internal pH are generally good measures for treatment effects, they were not always good measures (as tested with GLM) in the treatments with very low survival. Results should be interpreted carefully considering the small number of plants on which the measures are based.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Survival

Increased NH4+ concentrations resulted in decreased survival of S. pratensis and A. dioica (Fig. 1a,b; Table 2). At the lowest NH4+ concentration (10 µmol l−1), survival of S. pratensis and A. dioica ranged between 100 and 80%, while the survival of both species decreased rapidly at higher NH4+ concentrations to as low as 0% for S. pratensis at 500 µmol l−1. Although increased NH4+ concentrations resulted in slightly reduced survival of C. vulgaris and D. flexuosa, no significant NH4+ effect was observed (Fig. 1c,d; Table 2). The survival of S. pratensis and A. dioica was significantly affected by the pH of the growth medium at P < 0.001 (Fig. 1a,b; Table 2). Survival of these species was lower at low pH (average survival percentage of 38 and 60 for S. pratensis and A. dioica, respectively) compared with the survival at high pH (average survival percentage of 65 and 91 for S. pratensis and A. dioica, respectively). For both S. pratensis and A. dioica, an NH4+ × pH interaction effect was shown at the P < 0.1 level, suggesting enhanced detrimental effects of high NH4+ concentrations and low external pH.

image

Figure 1. Survival (%) over time for Succisa pratensis (a); Antennaria dioica (b); Calluna vulgaris (c); Deschampsia flexuosa (d). S. pratensis was grown at pH levels 4 and 5.5; A. dioica, C. vulgaris and D. flexuosa were grown at pH 3.5 and 5. NH4+ concentrations were applied as 10, 100, 500 and 1000 µmol l−1. Survival of G. pneumonanthe was 100% in all treatments (not shown).

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Table 2.  Statistical results of the effect of different NH4+ and pH levels on survival of Succisa pratensis, Antennaria dioica, Calluna vulgaris and Deschampsia flexuosa
SourcedfMean squareF
  1. Significance levels: +, P < 0.10; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Succisa pratensis
NH4 32.43 36.80***
pH 11.49 22.50***
NH4 × pH 30.19  2.92+
error240.07 
Antennaria dioica
NH4 30.35 21.53***
pH 11.81113.05***
NH4 × pH 30.05  2.98+
error240.02 
Calluna vulgaris
NH4 30.05  0.99
pH 10.00  0.03
NH4 × pH 30.12  2.34+
error240.05 
Deschampsia flexuosa
NH4 30.03  0.60
pH 10.00  0.00
NH4 × pH 30.04  0.77
error240.05 

No significant external pH effects on survival of C. vulgaris, D. flexuosa and G. pneumonanthe were observed. Although no significant effects of NH4+ or pH on survival of C. vulgaris were found, an interacting effect between NH4+ and pH on survival was found at the P < 0.1 level (Fig. 1c; Table 2). The effect of NH4+ and pH on survival of G. pneumonanthe was not tested as no mortality was observed during the experiment (data not shown).

Biomass

Both shoot and root biomass varied at different NH4+ concentrations and pH levels (Fig. 2a–e; Table 3). Gentiana pneumonanthe and D. flexuosa showed increased shoot biomass with increasing NH4+ concentrations at both pH levels. Root biomass of G. pneumonanthe and D. flexuosa did not differ significantly between different NH4+ levels. At pH 5, shoot and root biomass of A. dioica increased with increasing NH4+ levels from 10 up to 100 µmol l−1, whereas higher NH4+ levels (500 and 1000 µmol l−1) resulted in lower biomass. This indicated that A. dioica performed best at an NH4+ concentration of 100 µmol l−1. Below this concentration A. dioica was limited by N, and above 100 µmol l−1 the biomass of shoots and roots was negatively affected by high NH4+ concentrations. Similar patterns, showing optimal NH4+ concentrations for biomass production of shoots, were detected at both pH levels for S. pratensis and at pH 3.5 for C. vulgaris. At pH 5.5, NH4+ concentrations up to 500 µmol l−1 resulted in increased shoot biomass of S. pratensis (up to 0.2 g d. wt; Fig. 2b; Table 3) and lower (0.01 g d. wt) shoot biomass was observed at 1000 µmol l−1 NH4+. At the low pH levels, the highest shoot biomass for C. vulgaris (0.2 g d. wt) was measured at 500 µmol l−1 NH4+ and at 100 µmol l−1 for S. pratensis (0.08 g d. wt). However, the biomass of S. pratensis of the plants that survived at pH 4 was low at 500 and 1000 µmol l−1 NH4+ (Fig. 1a), and results shown in Fig. 2b should therefore be interpreted carefully.

image

Figure 2. Biomass of shoots and roots of Gentiana pneumonanthe (a); Succisa pratensis (b); Antennaria dioica (c); Calluna vulgaris (d); Deschampsia flexuosa (e), at the end of the experimental period. Plants were pooled together per container to determine biomass. Shoots are indicated by grey bars; roots by black bars. NH4+ concentrations were applied as 10, 100, 500 and 1000 µmol l−1. G. pneumonanthe and S. pratensis were grown at pH levels 4 and 5.5; A. dioica, C. vulgaris and D. flexuosa were grown at pH 3.5 and 5.

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Table 3.  Statistical results of the effect of different NH4+ and pH levels on biomass of Gentiana pneumonanthe, Succisa pratensis, Antennaria dioica, Calluna vulgaris and Deschampsia flexuosa
SourceShootRoot
dfMean squareFPdfMean squareFP
  • Only between-subjects effects are shown.

  • Significant P values (at α < 0.05) are indicated in bold.

Gentiana pneumonanthe
NH4 3 0.02 21.750.000 3 0.00  2.050.133
pH 1 0.04 46.270.000 1 0.01 20.150.000
NH4 × pH 3 0.00  3.030.049 3 0.00  1.050.389
error24 0.00  24 0.00  
Succisa pratensis
NH4 3 0.02  4.250.025 3 0.01  3.020.065
pH 1 0.01  0.980.338 1 0.00  0.260.618
NH4 × pH 2 0.00  0.510.610 1 0.00  0.000.983
error14 0.01  14 0.00  
Antennaria dioica
NH4 3 2.83  5.870.004 3 0.96  6.450.003
pH 150.47104.530.000 120.62138.460.000
NH4 × pH 3 1.46  3.020.050 3 0.75  5.020.008
error23 0.48  23 0.15  
Calluna vulgaris
NH4 3 9.86 11.160.000 3 4.45  1.080.376
pH 1 2.75  3.110.091 1 4.46  1.080.308
NH4 × pH 3 2.50  2.830.060 3 8.01  1.950.149
error24 0.88  24 4.12  
Deschampsia flexuosa
NH4 310.47  9.980.000 3 0.27  1.670.199
pH 1 0.29  0.270.606 1 0.02  0.140.709
NH4 × pH 3 1.93  1.840.167 3 0.04  0.240.870
error24 1.05  24 0.16  

The external pH of the medium markedly affected the biomass of the shoots and roots of G. pneumonanthe and A. dioica. At low pH levels, shoot and root biomass were on average 44 and 80% lower for G. pneumonanthe and A. dioica, respectively, as compared with the biomass at the higher pH levels (Fig. 2a,c). A negative but nonsignificant trend between biomass production and decreasing pH was visible for S. pratensis (Fig. 2b; Table 3). A significant interaction effect between NH4+ and pH of the growth medium on the root biomass was found for A. dioica, suggesting that negative pH effects were enhanced at high NH4+ concentrations.

Internal pH

Internal pH levels varied widely between the plant species and treatments, ranging from almost 6.5 to 4 (Fig. 3). The pH of the roots of G. pneumonanthe (average pH 4.27) was significantly lower compared with the pH of shoots (average of pH 5.04; one-way anova, MS = 9.379, df = 1, F = 205.331, P < 0.001; data not shown), whereas no significant differences between shoot and root pH of the other species were observed. In general, increasing NH4+ concentrations decreased internal pH levels significantly in both root and shoot material (Fig. 3; Table 4). Only in the roots of A. dioica in the pH 3.5 treatment was there a (nonsignificant) trend of a slightly higher internal pH at high NH4+ concentrations (One-way anova, MS = 0.151, df = 3, F = 0.810, P = 0.514).

image

Figure 3. Internal pH values of shoots and roots of Gentiana pneumonanthe (a); Succisa pratensis (b); Antennaria dioica (c); Calluna vulgaris (d); Deschampsia flexuosa (e). Shoots are indicated by grey bars; roots by black bars. NH4+ concentrations were applied as 10, 100, 500 and 1000 µmol l−1. G. pneumonanthe and S. pratensis were grown at pH levels 4 and 5.5; A. dioica, C. vulgaris and D. flexuosa were grown at pH 3.5 and 5.

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Table 4.  Statistical results of the effect of different ammonium (NH4+) and pH levels on internal pH of Gentiana pneumonanthe, Succisa pratensis, Antennaria dioica, Calluna vulgaris and Deschampsia flexuosa
SourceShootRoot
dfMean squareFPdfMean squareFP
  • Only the between-subjects effects were shown.

  • Significant P values (at α < 0.05) are indicated in bold.

Gentiana pneumonanthe
NH4 3 0.38  4.900.009 30.5037.880.000
pH 1 0.10  1.300.266 10.00 0.020.879
NH4 × pH 3 0.00  0.050.986 30.02 1.600.216
error24 0.08  240.01  
Succisa pratensis
NH4 3 0.66 21.420.000 30.7825.090.000
pH 1 0.00  0.160.693 10.15 4.660.050
NH4 × pH 2 0.05  1.470.263 20.09 2.990.086
error14 0.03  130.03  
Antennaria dioica
NH4 3 0.93 13.530.000 30.49 3.840.021
pH 113.23193.250.000 19.4773.840.000
NH4 × pH 3 0.11  1.580.219 31.3710.650.000
error26 0.07  260.13  
Calluna vulgaris
NH4 3 0.06  1.710.189 30.14 0.850.479
pH 1 0.00  0.010.919 10.01 0.030.860
NH4 × pH 3 0.02  0.530.664 30.38 2.330.097
error27 0.04  270.16  
Deschampsia flexuosa
NH4 3 0.34  2.830.057 32.3917.470.000
pH 1 0.28  2.320.139 10.08 0.580.451
NH4 × pH 3 0.23  1.940.148 30.41 3.010.048
error27 0.12  270.14  

Acidity of the growth medium significantly influenced only the internal pH of shoots and roots of A. dioica. At an external pH of 3.5, the pH of shoots and roots was 4.7 on average, whereas the pH of shoots and roots ranged between 5 and 6.5 when grown in a medium of pH 5 (average 5.9; Fig. 3c). Internal pH of shoots and roots of S. pratensis at 500 µmol l−1 NH4+ in the pH 5.5 treatment was low (4.5 and 4.3, respectively), and did not differ significantly from the pH levels at 1000 µmol l−1 NH4+ (4.8 and 4.7, respectively). As all plants had died in the treatments with 500 µmol l−1 NH4+ at pH 4, there were no data for this treatment. Results for the internal pH of plants grown in 1000 µmol l−1 NH4+ at pH 4 should be interpreted carefully, as these were based on a limited number of plants.

The pH of the external medium enhanced the effects of the NH4+ concentrations for A. dioica and D. flexuosa, illustrated by significant NH4+ × pH interaction effects (Table 4). The internal pH of A. dioica and D. flexuosa showed a decrease at pH 5 with increasing NH4+ concentrations, whereas at pH 3.5 no significant NH4+ effects were measured (Fig. 3c,e). Thus the effects of NH4+ on internal pH differed significantly between pH levels, which explains the interaction effect shown in Table 4.

Nutrient composition and C/N content

Increased NH4+ concentrations resulted in a significant decrease in Ca and Mn concentrations in both shoots and roots of G. pneumonanthe and D. flexuosa, and in shoots of C. vulgaris (Table 5). Concentrations of K also decreased with increasing NH4+ concentration, except for shoots of G. pneumonanthe in which the concentrations increased. In C. vulgaris, K decreased in the roots with increasing NH4+ (from an average of 98 µmol g−1 d. wt for both pH levels to 75 µmol g−1 d. wt), but showed an optimum curve with increasing NH4+ in the shoots. The K concentration was significantly higher in the 100 and 500 µmol l−1 NH4+ treatments (average 237 µmol g−1 d. wt for both pH levels) compared with the treatments with 10 or 1000 µmol l−1 NH4+ (average 183 µmol g−1 d. wt for both pH levels). Concentrations of Mg decreased with increasing NH4+ concentration only in roots of G. pneumonanthe, C. vulgaris and D. flexuosa. There were no differences measured in the shoots of these three species. Other negative effects of NH4+ concentration could be found for P in shoots of C. vulgaris (lowest value 87 µmol g−1 d. wt in the 1000 µmol l−1 NH4+, pH 3.5 treatment) and roots of G. pneumonanthe (50 µmol g−1 d. wt in the 1000 µmol l−1 NH4+, pH 3.5 treatment). A significant increase in S concentration with increasing NH4+ concentrations was found in the shoots of G. pneumonanthe, C. vulgaris and D. flexuosa and in the roots of D. flexuosa. Nitrogen content of all tissues increased significantly with increasing NH4+ concentrations in all species, except for shoots of G. pneumonanthe. In D. flexuosa tissue, and in the roots of G. pneumonanthe, an increase in C content was measured with increasing NH4+ concentrations.

Table 5.  Statistical results of a general linear model (GLM) procedure on nutrient content of Calluna vulgaris, Deschampsia flexuosa and Gentiana pneumonanthe with ammonium and pH levels as fixed factors
SpeciesRootShoot
CaMgKMnSPCNCaMgKMnSPCN
  1. Results for Antennaria dioica and Succisa pratensis could not be shown as the results did not allow for statistical analysis because of high mortality and lack of material for the analysis.

  2. Significance levels: ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. Effects are indicated between brackets: (+) positive effect; (–) negative effect; (opt) optimum with lowest concentrations in 10 or 1000 µmol l−1 NH4+ treatments and higher concentrations in the 100 and 500 µmol l−1 NH4+ treatments.

Calluna vulgaris
pHnsnsns** (+)nsnsnsns* (+)* (+)* (+)* (+)* (+)nsns* (opt)
NH4ns* (–)** (–)*** (–)nsnsns*** (+)** (–)ns*** (opt)** (–)*** (+)* (–)ns*** (+)
pH × NH4nsns*nsnsnsnsnsnsnsnsnsnsnsns*
Deschampsia flexuosa
pHnsnsnsnsnsnsns* (–)nsnsnsnsnsnsnsns
NH4* (–)* (–)* (–)*** (–)*** (+)ns* (+)** (+)*** (–)ns** (–)*** (–)*** (+)ns*** (+)*** (+)
pH × NH4nsnsnsnsnsnsnsnsnsnsnsnsnsnsnsns
Gentiana pneumonanthe
pH*** (+)*** (+)*** (+)*** (+)*** (+)*** (+)** (–)ns*** (+)*** (+)*** (+)*** (+)*** (+)*** (+)** (–)ns
NH4*** (–)** (–)*** (–)*** (–)ns** (–)* (+)* (+)** (–)ns** (+)** (–)*** (+)nsnsns
pH × NH4nsnsnsnsnsnsnsnsnsnsnsnsnsnsnsns

The pH level of the treatments did not affect D. flexuosa, except for the N content of the roots. In contrast, higher values of almost all nutrients were found at the highest pH level (Table 5) in shoots of C. vulgaris and in both shoots and roots of G. pneumonanthe. Concentrations of Mn were also higher at the higher pH level in the roots of C. vulgaris. Carbon content in both shoots and roots of G. pneumonanthe was significantly lower at pH 4. A pH × NH4+ interaction effect was measured for K concentration in the roots of C. vulgaris and for the N content of the shoots.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Effects of NH4+ concentrations and pH on the survival and growth of heathland species

Antennaria dioica and S. pratensis were expected to be acid-sensitive species. Acid-sensitive species are naturally found on slightly buffered soils (pH 4.5–6) where mineral N is available in the form of nitrate (NO3) as well as in the form of NH4+. These species will be greatly affected by increased NH4+ concentrations, as they prefer NO3 over NH4+ (Falkengren-Grerup & Lakkenborg-Kristensen, 1994; Falkengren-Grerup, 1995). Our results support the hypothesis that A. dioica and S. pratensis are acid-sensitive herbaceous species suffering from combined effects of high NH4+ concentrations and low pH. NH4+ toxicity was observed for A. dioica and S. pratensis at pH levels that resembled weakly buffered soil conditions (pH 5 and 5.5 treatments). These negative effects of NH4+ nutrition on survival and growth of S. pratensis and on survival of A. dioica were even more pronounced at pH levels resembling acidified soil conditions (pH 3.5 and 4 treatments), as illustrated by a slight NH4+ × pH interaction effect (Table 2).

Biomass of A. dioica was largely determined by pH of the medium (Fig. 2), with a lower total biomass in the acidic treatment. No effect of NH4+ on root biomass was observed at pH 3.5, as opposed to a significant negative NH4+ effect at pH 5, which is illustrated by the NH4+ × pH interaction effect (Table 3). This suggests that growth of A. dioica is affected by NH4+ concentrations at the higher pH levels, whereas at pH 3.5 growth conditions are detrimental, irrespective of NH4+ levels.

In contrast, the species that were classified as acid tolerant species, D. flexuosa and C. vulgaris, demonstrated an increased total biomass with increasing NH4+ concentrations. Only at low pH did C. vulgaris suffer from very high NH4+ concentrations (1000 µmol l−1), indicated by a lower biomass and a slight NH4+ × pH interaction effect on survival. This shows that strongly increased NH4+ concentrations (eutrophication) in combination with low pH (acidification) might well affect the performance of C. vulgaris. An increased biomass of both acid-tolerant species with increasing NH4+ concentrations suggested N limitation at the lower NH4+ concentrations.

The herbaceous species G. pneumonanthe was expected to be slightly acid-tolerant. This was illustrated by an increase in biomass and survival of G. pneumonanthe with increasing NH4+ concentrations. The species was able to withstand low pH in combination with high NH4+ concentrations. At high NH4+ concentrations (500 and 1000 µmol l−1), biomass did not either increase or decrease, indicating that other nutrients may have become limiting.

Calluna vulgaris, D. flexuosa and G. pneumonanthe are naturally found on slightly acidic to more acidic soils, and might be adapted to NH4+ nutrition as NH4+ is the common form of inorganic N at low soil pH (Gigon & Rorison, 1972; Troelstra et al., 1990). This might explain why these species, apart from only C. vulgaris at low pH, are not affected by high NH4+ concentrations. Data from several studies have shown that acid-tolerant species can tolerate high NH4+ concentrations (Gigon & Rorison, 1972; Falkengren-Grerup & Lakkenborg-Kristensen, 1994; Falkengren-Grerup, 1995).

Although survival of G. pneumonanthe did not differ from that of C. vulgaris and D. flexuosa in different NH4+ and pH treatments, negative effects of acidification on biomass were observed. Based on field data, G. pneumonanthe can be regarded as more sensitive to acidic conditions than C. vulgaris and D. flexuosa. As there were no differences in survival in this study, this sensitivity may well be caused by indirect pH effects via increased Al toxicity. From earlier studies it is known that increased acidification results in increased Al concentrations, which can be toxic for many herbaceous species (Kinraide, 1997; van den Berg et al., 2003). In this experiment, plants were grown on hydroculture in which Al concentrations were kept constantly low. The results of this study therefore do not allow us to draw conclusions concerning indirect pH effects, such as Al toxicity.

Internal pH differences and nutrient content

Many studies have reported on the mechanisms for NH4+ toxicity (for a review see Britto & Kronzucker, 2002). One of these mechanisms is the decrease of the internal pH of the plant at elevated NH4+ concentrations in the rhizosphere (Raven, 1986; Marschner, 1995). In the present study a decrease in internal pH at increasing NH4+ concentrations was observed for most species in both shoots and roots, which is in accordance with other studies that found proton production when NH4+ is assimilated (Raven & Smith, 1976; Raven, 1986; Gerendás et al., 1990; Britto & Kronzucker, 2002). The proton production results in internal acidification, and protons have to be excreted or neutralized to maintain cell pH homeostasis. This leads to acidification of the rhizosphere (Findenegg, 1987; Van Beusichem et al., 1988; Goodchild & Givan, 1990; De Graaf et al., 2000).

In this study we found a significant pH effect and NH4+× pH interaction effect on the internal pH of roots of the acid-sensitive A. dioica (Table 4), but not of S. pratensis. For the latter species, the effect of a low pH was too dominant (Fig. 2). The effects of decreasing pH and increasing NH4+ in the medium on the internal pH of A. dioica were visible in both shoots and roots. The NH4+ × pH interaction effect on internal pH of the roots and shoots illustrates the sensitivity to low pH levels and increasing NH4+ concentrations. These effects resemble those found for the biomass of A. dioica (Fig. 2; Table 3).

Surprisingly, internal pH values decreased with increasing NH4+ concentrations also for the acid-tolerant G. pneumonanthe and D. flexuosa, despite the fact that survival and biomass increased (Tables 2 and 3). These species appeared to be tolerant to low internal pH levels, suggesting that internal pH may not be a suitable indicator for NH4+ toxicity and/or plant performance. This is consistent with an earlier study by Paulissen et al. (2004), who did not find any serious physiological problems for Polytrichum commune when internal pH decreased due to NH4+ nutrition.

Increased NH4+ concentrations resulted in an increase in N concentration in all tissues of all species except for the shoots of G. pneumonanthe. This indicates an increased N uptake with increasing NH4+ in the treatments for these species. In addition, N : P ratios in all species were between 0 and 3 g g−1 and remained well below the suggested minimal value for P limitation (Koerselman & Meuleman, 1996). NH4+ uptake resulted in a decrease in cation uptake (Table 5), which is known to be a possible NH4+ toxicity mechanism as it can result in cation deficiency (Kirkby, 1968; Van Beusichem et al., 1988). In addition, a study by De Graaf et al. (1998) showed Ca and Mg deficiency, caused by reduced uptake, in C. dissectum grown at high NH4+ concentrations and low pH. Lucassen et al. (2003) found similar results in media with both high and low pH, and concluded that NH4+ was responsible for the reduced cation content. Cation concentration (Ca, Mg, K, Mn) of both acid-tolerant species (C. vulgaris and D. flexuosa) and the supposed slightly acid-tolerant G. pneumonanthe was generally reduced at high levels of NH4+. However, these effects did not lead to a reduced performance; on the contrary, the biomass of these three species increased with increasing NH4+. Only in the shoots of C. vulgaris treated with high NH4+ concentrations were indications of chlorosis found, indicated by a light green colour (data not shown). For G. pneumonanthe and D. flexuosa no indications of cation deficiency could be noted.

The concentration of S in the shoots of G. pneumonanthe, C. vulgaris and D. flexuosa increased with increasing NH4+ concentrations. This is in agreement with studies by Kirkby (1968) and van Beusichem et al. (1988), who found an increased uptake of anions including sulfate (SO42–) with increasing NH4+ nutrition. This enhanced uptake was attributed to the replacement of NO3 reduction with SO42– reduction, which is necessary for the regulation of the pH-stat mechanisms when NO3 becomes limiting.

Next to the effects of NH4+, a decrease in external pH resulted in a decreased uptake of cations and reduced C concentration in the roots and shoots of G. pneumonanthe. This effect was also visible for the cation concentrations in the shoots of C. vulgaris. These results are consistent with earlier studies in which a decrease in the pH of the rhizosphere and apoplast was found to decrease cation uptake, resulting in cation deficiencies of the plant (Van Beusichem et al., 1988; Boxman et al., 1991; Marschner, 1995). Clearly, the acid-tolerant species D. flexuosa was not affected by the external pH level apart from the N concentration in the roots. Shoot tissue of C. vulgaris, however, also supposed to be acid-tolerant, contained more cations at the high pH level compared with the tissue at low pH level. As mentioned above, C. vulgaris did not show negative effects on survival or biomass related to external pH, which suggests that the effect of external pH on nutrient content did not lead to cation deficiencies. However, the combination of a low pH and very high NH4+ concentration might lead to serious deficiencies in time. Shoots of C. vulgaris grown at 1000 µmol l−1 NH4+ showed indications of chlorosis (data not shown). As C. vulgaris responded slowly to subjected treatments, the experimental period of this study might be too short to record these effects; such effects may, however, have a significant impact in latter stages.

Lack of material caused by high mortality and poor growth prevented us from analysing S. pratensis plants for nutrient content. It is, however, likely that cation deficiency was responsible for the decline of this species on high NH4+ and low pH medium. Such effects were shown earlier for C. dissectum (De Graaf et al., 1998).

Implications for biodiversity in heathlands

Plant diversity is rapidly declining in heathlands and matgrass swards in Western Europe, and this may be the result of increased N deposition, which is causing acidification of the soil (Roelofs, 1986; Aerts et al., 1990; Bobbink et al., 1998; Lee & Caporn, 1998; Krupa, 2003). In earlier studies it was found that high NH4+ concentrations are detrimental for germination, growth and survival of several (rare) heathland species (De Graaf et al., 1998; Dorland et al., 2003), and these effects may be exacerbated at low soil pH (Lucassen et al., 2003). In degraded acidified heathlands, soil solution pH ranged between 3.8 and 4.5, and NH4+ concentrations ranged between 300 and 650 µmol kg−1 d. wt (De Graaf et al., 1994; Roelofs et al., 1996; Dorland et al., 2003). It was expected that the presence and decline of species in heathlands was correlated with the ranges of both NH4+ and pH. In this study we were interested in the specific interaction effects between NH4+ and pH on the performance of different plant species. In order to test this, we used a hydroponic approach with NH4+ and pH values comparable with those found in the field. Hydroponic studies provide a useful tool for investigating plant responses to specific dose-dependent factors such as the NH4+ concentration and pH of the medium. However, these types of experiment do not fully reflect the situations that can be found in the field. For instance, with hydroponic studies the potential role of mycorrhiza, assimilation of nutrients via shoots and uptake kinetics are not included. Also, the concentration of all elements in the growth medium is kept constant, which does not resemble realistic field situations. However, hydroponic techniques allowed us to determine the plant responses at different, controlled NH4+ concentrations and pH levels in a direct and straightforward manner, including measuring the interaction of both independent variables.

The results of this study show that NH4+ concentration and pH of the solution can have strong determining and interacting effects on vegetation. These variables might also affect the composition of heathlands, with the reason for the decline varying for each species. The decline of A. dioica from heathlands may be explained by negative effects of acidification and/or NH4+ toxicity at low pH and by NH4+ toxicity at high pH levels, whereas the decline of G. pneumonanthe in the Netherlands may be caused by factors other than NH4+ toxicity, as no detrimental effects of increased NH4+ concentrations on the performance of G. pneumonanthe were observed. Although this species is regarded as acid-tolerant, indications for sensitivity to acidification were found. Other factors associated with low soil pH, such as high Al concentrations, might also be important. The results described in this study are in accordance with other studies that attributed the strong decline of many plant species of dry and wet heathlands and matgrass swards to both eutrophication and acidification (Roelofs, 1986; Aerts et al., 1990; Lee & Caporn, 1998; Krupa, 2003). From our results, we may suggest that NH4+ toxicity can seriously affect plant diversity in acidified heathlands.

In contrast, grasses such as D. flexuosa can benefit from these soil conditions. The results show that this species can increase biomass at high concentrations of NH4+ nutrition, enabling this species to outcompete slower-growing species. Therefore competition for light and nutrients with grasses such as D. flexuosa may explain the strong decline in abundance and number of many herbaceous species.

Current restoration measures involve liming of the soil in order to reduce the effects of acidification. Liming results in increased soil pH and buffering cations (Dorland et al., 2004), thereby reducing direct negative acidity effects on plants. In addition, Feng et al. (1992) found that proton release from the roots could be stimulated in an acid environment by the addition of Ca. Therefore liming also reduces NH4+ toxicity symptoms resulting from decreased internal pH. In order to conserve acid-sensitive, herbaceous species in heathlands, restoration measures such as liming and removal of grasses by grazing and/or sod-cutting are a prerequisite.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank the Netherlands Organization for Scientific Research (NWO) for financially supporting our work (Grant no. 014.22.061 and 014.22.062). Gerrit Rouwenhorst, Paul van der Ven, Germa Verheggen and Roy Peters are thanked for their analytical help. We also appreciate the valuable comments of Leon Lamers, Jos Verhoeven and Dennis Whigham on an earlier draft of this manuscript.

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  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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